We are studying the reactions required for the replication of duplex DNA in vitro using proteins encoded by T4 bacteriophage. The T4 gene 41 and t4 gene 61 proteins synthesize ribopentanucleotides of sequence pppACNNN which are elongated by the T4 gene 43 DNA polymerase, T4 gene 45 protein, the complex of the T4 gene 44 and T4 gene 62 proteins (a DNA-dependent ATPase), and the t4 gene 32 helix destabilizing protein. The 44/62 and 45 proteins enable the polymerase to reach and pass through hairpin barriers in single-stranded templates and permit the polymerase to bind more tightly to DNA. 32 Protein eliminates barriers in single-stranded templates and is required, together with 43, 44/62, and 45 for synthesis on duplex templates. We are investigating the mechanism of primer synthesis and the DNA unwinding (helicase) activities associated with these T4 proteins. We are searching for T4 or host proteins required for the initiation of replication of duplex T4 DNA. We are characterizing a restriction nuclease, controlled by a late T4 protein, which cuts duplex DNA at specific sites. Amino acid sequencing of the 45 and 44/62 proteins is in progress.